1. Field of the Invention
The present invention relates to a polypeptide having ceramidase activity, and a gene encoding said polypeptide. More particularly, the present invention relates to an amino acid sequence of ceramidase as well as a nucleotide sequence encoding the amino acid sequence which are useful as an reagent for lipid engineering for analyzing structure and action of ceramide and as an index for diagnosis of atopic dermatitis. In addition, the present invention relates to a method for producing a polypeptide having ceramidase activity by gene engineering. Further, the present invention relates to a method for detecting the polypeptide, and a method for detecting a gene encoding the polypeptide. Further, the present invention relates to a method for detecting atopic dermatitis by utilizing the above method for detecting the polypeptide and the above method for detecting the gene.
2. Discussion of the Related Art
Ceramidase is an enzyme that hydrolyses ceramide, which is one of sphingolipids, into sphingosine and a fatty acid. Sphingosine formed by hydrolysis of ceramide by ceramidase has various physiological activities, such as inhibition of protein kinase C, activation of phospholipase D, inhibition of a calmodulin-dependent enzyme, and the like, and is an important substance which is considered to have a role in regulating cellular functions through its involvement in cell proliferation and intracellular signal transduction. The regulation of the level of such sphingosine is an important role played by ceramidase.
Ceramidases are classified on the basis of its optimum pH into an acidic ceramidase and a neutral/alkaline ceramidase. There have been reported that ceramidases having optimum pH in an acidic region are present in mammalian tissues such as rat brain [Biochemistry, 8, 1692-1698 (1969)], guinea pig epithelial cell [J. Biol. Chem., 270, 12677-12684 (1995)], human kidney [Biochim. Biophys. Acta, 398, 125-131 (1975)], spleen [Biochim. Biophys. Acta, 1004, 245-251 (1989)], fibroblast [Biochem. J., 205, 419-425 (1982)] and epithelium [FEBS Lett., 268, 110-112 (1990)]; human urine [J. Biol. Chem., 270, 11098-11102 (1995)], and the like.
Among these ceramidases, the amino acid sequences and the nucleotide sequences of ceramidase purified from human urine have been determined [J. Biol. Chem., 271, 33110-33115 (1996)]. Utilizing the homology with this gene, a ceramidase gene of a mouse has also been obtained [Genomics, 50, 267-274 (1998)]. However, all these are acidic ceramidases derived from mammals, and no amino acid sequences or nucleotide sequences of neutral/alkaline ceramidase or of ceramidases derived from microorganisms have yet been determined.
On the other hand, a ceramidase-producing microorganism is known to be present on the epidermis of lesion of atopic dermatitis, and this enzyme is suggested to be causative of or be involved in the exacerbation of atopic dermatitis. However, this enzyme is ceramidase of which optimum pH is within an alkaline region (hereinafter referred to as alkaline ceramidase) [J. Biol. Chem., 273, 14368-14373 (1998)].
When a naturally-occurring ceramidase is produced from a ceramidase-producing microorganism, it is necessary to add an expensive sphingolipid to a culture medium in order to induce the production of an enzyme, and in purification step, it is necessary to separate and remove the remaining sphingolipid or a degradation product thereof from a fraction containing ceramidase. In addition, since enzymes other than ceramidases such as sphingomyelinase are simultaneously produced during the culture, it is difficult to isolate and purify only a desired ceramidase from these enzymes. Moreover, the amino acid sequences or the gene structures of microorganism-derived alkaline ceramidase are completely unknown, so that the method for producing ceramidase by gene engineering cannot be utilized.
Furthermore, although the presence of the ceramidase-producing microorganism is expected to be used as an index for diagnosing atopic dermatitis and as a method for confirming a therapeutic effect, no means for simply detecting or identifying such a microorganism has yet been known. Conventional methods have necessitated extremely complicated processes that the microorganism is isolated and then cultured, and thereafter its ceramidase activity is assayed. Thus, a method for producing a high-purity ceramidase derived from microorganisms at a lower cost is desired as well as a method for detecting a ceramidase-producing microorganism in a simple, non-time-consuming procedure.
A first object of the present invention is to provide a polypeptide having ceramidase activity. A second object of the present invention is to provide a gene encoding the above polypeptide. A third object of the present invention is to provide a transformant harbouring the above gene. A fourth object of the present invention is to provide a production method capable of easily and massively producing high-purity ceramidase utilizing the above transformant. A fifth object of the present invention is to provide an oligonucleotide probe or a primer capable of specifically hybridizing with the gene of the present invention. A sixth object of the present invention is to provide an antibody or a fragment thereof, which is capable of specifically binding to a polypeptide of the present invention. A seventh object of the present invention is to provide a method for detecting a ceramidase gene by using the oligonucleotide probe or the primer of the present invention. An eighth object of the present invention is to provide a method for detecting ceramidase by using the antibody or the fragment thereof of the present invention, and a kit used therefor. Furthermore, a ninth object of the present invention is to provide a method for detecting atopic dermatitis by utilizing a method for detecting the ceramidase gene or method for detecting ceramidase of the present invention.
As a result of intensive investigation in order to isolate a gene encoding a polypeptide having ceramidase activity, the present inventors have succeeded in isolation of a gene encoding a polypeptide having ceramidase activity and elucidation of the nucleotide sequence of the gene. In addition, based on such findings, the present inventors have established a method capable of easily and massively producing high-purity ceramidase; a method for detecting a polypeptide having ceramidase activity; and a method for detecting the ceramidase gene, and the present invention has been completed thereby. In addition, the correlation between such ceramidase and atopic dermatitis has been found, whereby a method for simply detecting atopic dermatitis has been able to be established.
Specifically, the gist of the present invention follows:
[1] a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1 in Sequence Listing, or a polypeptide having an amino acid sequence which has substitution, deletion, addition or insertion of one or more amino acids in the amino acid sequence of SEQ ID NO: 1 and having ceramidase activity;
[2] a gene encoding the polypeptide of the above item [1];
[3] the gene according to the above item [2], wherein the gene has a nucleotide sequence as shown in SEQ ID NO: 2 in Sequence Listing, or a nucleotide sequence which has substitution, deletion, addition or insertion of one or more bases in the nucleotide sequence of SEQ ID NO: 2 and encodes a polypeptide having ceramidase activity;
[4] a gene capable of hybridizing with the gene of the above item [2] under stringent conditions, and encoding a polypeptide having ceramidase activity;
[5] a transformant harbouring the gene of the above item [2];
[6] a method for producing a polypeptide having ceramidase activity, comprising culturing the transformant of the above item [5], and collecting a polypeptide having ceramidase activity from the resulting culture;
[7] an oligonucleotide probe or a primer, which is capable of hybridizing under stringent conditions with the gene of the above item [2] or with a gene having a nucleotide sequence complementary thereto;
[8] a method for detecting a gene encoding a polypeptide having ceramidase activity, by using the oligonucleotide probe and/or the primer of the above item [7];
[9] a kit for detection of a gene encoding a polypeptide having ceramidase activity, comprising the oligonucleotide probe and/or the primer of the above item [7];
[10] an antibody or a fragment thereof, which is capable of specifically binding to the polypeptide of the above item [1];
[11] a method for detecting a polypeptide having ceramidase activity, by using the antibody or the fragment thereof of the above item [10];
[12] a kit for detection of a polypeptide having ceramidase activity, comprising the antibody or the fragment thereof of the above item [10];
[13] a method for detecting atopic dermatitis, comprising detecting a gene encoding a polypeptide having ceramidase activity by the method of the above item [8]; and
[14] a method for detecting atopic dermatitis, comprising detecting a polypeptide having ceramidase activity by the method of the above item [11].